ABSTRACT
Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+. Catalysis was associated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A7C7ATATAGCGCGT2, as well as a preference for cleaving within CG-rich regions of dsDNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg2+ coordination site composed of the L32Tyr and L27dHis side chains and a DNA 3'-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V(H) and V(L) genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important-rarely examined-function for some antibody molecules.
Subject(s)
Antibodies, Antinuclear/metabolism , Antibodies, Catalytic/metabolism , Antibodies, Monoclonal/metabolism , DNA/immunology , DNA/metabolism , Animals , Antibodies, Antinuclear/chemistry , Antibodies, Antinuclear/genetics , Antibodies, Catalytic/chemistry , Antibodies, Catalytic/genetics , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Base Sequence , Binding Sites , DNA/chemistry , Hydrolysis , In Vitro Techniques , Kinetics , Mice , Models, Molecular , Mutation , Protein ConformationABSTRACT
Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+ ions. DNA hydrolyzing activity was associated with BV 04-01 IgG, Fab, and SCA 04-01 proteins. Pronounced cleavage specificity for both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of the oligonucleotide A7C7ATATAGCGCGT7 as well as preference for cleavage within CG-rich regions of double-stranded DNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA 04-01 and two SCA 04-01 mutants (L32Phe and L27dHis) were used to model the catalytically active antibody site utilizing the previously resolved X-ray structure of (dT)3 liganded Fab 04-01. The resulting model suggested that BV 04-01 activates the target phosphodiester bond by induction of conformational strain. In addition, the antibody-DNA complex contained a potential Mg2+ ion coordination site composed of the L32Tyr and L27dHis amino acid side chains and a DNA 3'-phosphodiester group. Induction of strain and metal coordination could be constituents of a mechanism by which this antibody catalyzed DNA hydrolysis. Sequence data for BV 04-01 VH and VL genes suggested that the proposed catalytic antibody active site was germ-line encoded. This observation suggests the hypothesis that catalytic activity might represent an important but unspecified function of some antibody molecules.
Subject(s)
Antibodies, Catalytic/immunology , Antibodies, Monoclonal/immunology , Autoantibodies/immunology , DNA, Single-Stranded/immunology , DNA/metabolism , Animals , Binding Sites , Catalysis , Crystallography, X-Ray , Hydrolysis , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/immunology , Kinetics , Models, Molecular , Protein Conformation , Tumor Cells, CulturedSubject(s)
Antibodies, Antinuclear/metabolism , Antibodies, Catalytic/metabolism , Autoimmune Diseases/immunology , DNA/metabolism , Lupus Erythematosus, Systemic/immunology , Base Sequence , Deoxyribonuclease I/metabolism , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides/chemistry , PlasmidsABSTRACT
Catalysis by antibodies could be a frequent phenomenon if the immune system generates a sufficiently diverse number of antibody-active sites, some of which may possess catalytic activity. A catalytic antibody can be expected to do more damage than one that simply binds antigen. The best biochemical marker of systemic lupus erythematosus (SLE) is presence of autoantibodies to DNA. In the present article, we describe the DNA-hydrolyzing activity of DNA-binding autoantibodies purified from SLE patients. The substrates employed were supercoiled plasmid, radiolabeled plasmid fragments, and oligonucleotides. Hydrolysis of DNA by the antibodies was indicated by the appearance of fragments visualized by ethidium bromide staining of agarose gels or autoradiography of polyacrylamide gels. Changes in linear dichroism values were also indicative of DNA hydrolysis. The antibody activity was purified by protein A-sepharose chromatography, high-performance liquid chromatography gel filtration, and DNA-affinity chromatography. Scrupulous control studies were done to demonstrate that DNA-hydrolyzing activity really belongs to the antibodies. Purified Fab fragments showed hydrolyzing activity, whereas the Fc fragment was inactive. The specificity of DNA cleavage was investigated, and the rate parameters of hydrolysis by antibodies and conventional nucleases were compared.
Subject(s)
Antibodies, Catalytic/metabolism , Antibodies/metabolism , DNA/metabolism , Lupus Erythematosus, Systemic/immunology , Antibodies/immunology , Antibodies/isolation & purification , Antibodies, Catalytic/immunology , Antibodies, Catalytic/isolation & purification , Base Sequence , Biomarkers , Ethidium/chemistry , Humans , Hydrolysis , Immunoglobulin Fragments/immunology , Immunoglobulin Fragments/metabolism , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Immunoglobulin G/metabolism , Molecular Sequence DataABSTRACT
The multigene family of human Na,K-ATPase is composed of 5 alpha-subunit genes, 3 of which were shown to encode the functionally active alpha 1, alpha 2 and alpha 3 isoforms of the catalytic subunits. This report describes the isolation, mapping and partial sequencing of the fourth gene (ATP1AL1) that was demonstrated here to be functionally active and expressed in human brain and kidney. Limited DNA sequencing of the ATP1AL1 exons allowed one to suggest that the gene probably encodes a new ion transport ATPase rather than an isoform of the Na,K-ATPase or the closely related H,K-ATPase.
Subject(s)
Adenosine Triphosphatases/genetics , Multigene Family , Sodium-Potassium-Exchanging ATPase/genetics , Transcription, Genetic , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Base Sequence , Biological Transport , Brain/enzymology , Electrophoresis, Agar Gel , Exons , Humans , Introns , Kidney/enzymology , Molecular Sequence Data , Oligonucleotide Probes , Polymerase Chain Reaction , Sequence Homology, Nucleic AcidABSTRACT
Human brain cDNA libraries were screened with cDNA inserts corresponding to the mRNA for the Na+,K(+)-ATPase alpha-subunit from pig kidney. The results obtained demonstrate the existence of two highly homologous mRNAs encoding the alpha- and alpha III-isoforms of the Na+,K(+)-ATPase catalytic subunit.
